# Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

## TL;DR

This paper explains how using cycloheximide in ribosome profiling experiments can distort conclusions about how quickly ribosomes translate different codons in yeast.

## Contribution

The study identifies how cycloheximide alters codon-specific elongation rates, resolving contradictory findings in ribosome profiling.

## Key findings

- Experiments with cycloheximide show altered ribosome density patterns downstream of codons.
- Codons decoded by less abundant tRNAs were actually translated more slowly before cycloheximide was added.
- Conclusions from cycloheximide-based experiments may need reevaluation due to distorted translation dynamics.

## Abstract

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.